Surface measurement of in-vivo subjects using spot projector

Abstract

The invention provides for surface mapping of in-vivo imaging subjects using a single camera and an illuminator that projects a plurality of targets such as spots on the subject. By limiting the depth-of-field of the camera lens, or of the illuminator optics, or both, a spatial plane is defined in which the spots are most sharply in focus. Controlled displacement of this plane relative to the subject is achieved through movement of the mechanical stage on which a subject is placed; or through movement of the best-focus plane by adjustment of the camera, lens, or illuminator optics. Images are taken at several relative positions of the best-focus plane and the subject, and the height of individual points on the subject is determined through analysis of focus, given the known displacements. A mesh or other surface can be constructed from individual point locations, to provide a surface map of the subject. Accuracy of 0.5 mm can be readily attained for mice and similarly sized subjects.

1. A method for determining a contour of a subject using an in-vivo measurement system having a stage supporting the subject, control elements connected to a controller, and an imaging system having an optical axis, the method comprising the steps of:
 generating, by a projector, rays incident on the subject supported on the stage of the in-vivo measurement system for producing a light pattern on the subject, wherein the rays incident on the subject for producing the light pattern are directed substantially in the direction of the optical axis of the imaging system of the in-vivo measurement system; 
 incrementally moving, by the control elements, at least one of the stage and the object plane of the imaging system to a plurality of positions substantially in a direction of an optical axis of the imaging system; 
 taking, by the imaging system, images of the subject at each of the stage positions; 
 analyzing, by the controller, sharpness of focus for each of a plurality of points in the light pattern to determine the heights of each of the plurality of points; and 
 constructing, by the controller, a contour of the subject from the height values. 
 
     
     
       2. The method of  claim 1 , wherein the rays that produce the light pattern have a depth of field that is shorter than the depth of field of the imaging system. 
     
     
       3. The method of  claim 1 , wherein the rays that produce the light pattern have a depth of field that is larger than the depth of field of the imaging system. 
     
     
       4. The method of  claim 1 , wherein said step of constructing comprises constructing a mesh from a network of the individual height values as an estimate of an actual contour of the subject. 
     
     
       5. The method of  claim 1 , wherein the step of generating comprises reflecting the rays from a partially reflective mirror toward the subject. 
     
     
       6. The method of  claim 1 , wherein the subject is a mouse or a rat. 
     
     
       7. The method of  claim 1 , wherein said step of taking images is performed using reflected light. 
     
     
       8. The method of  claim 1 , further comprising the step of taking an in-vivo image of the subject for an in-vivo imaging experiment and analyzing the in-vivo image using the constructed contour. 
     
     
       9. The method of  claim 8 , wherein the in-vivo image is obtained using fluorescence or bioluminescence emitted from the subject. 
     
     
       10. The method of  claim 9 , wherein the step of taking images of the subject at each of the positions is performed using reflected ambient light. 
     
     
       11. The method of  claim 1 , wherein the pattern of light is a grid of spot targets. 
     
     
       12. An in-vivo imaging system, comprising:
 a stage for supporting a subject in the in-vivo measurement system; 
 an imaging system of the in-vivo measurement system having an optical axis; 
 a projector configured to generate rays incident on the subject for producing a light pattern on the subject, the rays incident on the subject directed substantially in the direction of the optical axis of the imaging system; and 
 control elements connected to a controller, the control elements configured to move at least one of the stage and imaging system toward or away from the other of the stage and imaging system, said controller and imaging system configured to determine a best-focus height of a plurality of points in the light pattern by analyzing sharpness of focus for each of the plurality of points, whereby a contour of the subject is determinable from the best-focus heights. 
 
     
     
       13. The in-vivo imaging system of  claim 12 , wherein said controller stores an executable program for determining the contour of the subject, the program comprising the executable steps of:
 activating said projector to produce the light pattern on the subject supported on said stage; 
 incrementally moving, by said control elements, at least one of said stage and said imaging system to a plurality of positions such that said stage and said imaging system are moved toward or away from each other; 
 taking, by said imaging system, images at each of the stage positions; 
 analyzing, by said controller, sharpness of focus for each of the plurality of points in the light pattern to determine the best-focus heights of each of the plurality of points; and 
 constructing, by the controller, the contour of the subject from the best-focus height values. 
 
     
     
       14. The in-vivo imaging system of  claim 13 , wherein the program further comprises the executable step of taking an in-vivo image of the subject for an in-vivo imaging experiment and analyzing the in-vivo image using the constructed contour. 
     
     
       15. The in-vivo imaging system of  claim 14 , wherein the in-vivo image is obtained using fluorescence or bioluminescence emitted from the subject. 
     
     
       16. The in-vivo imaging system of  claim 15 , wherein the step of taking images of the subject at each of the positions is performed using reflected ambient light. 
     
     
       17. The in-vivo imaging system of  claim 12 , wherein said projector has an illumination device and a grid for forming the light pattern. 
     
     
       18. The in-vivo imaging system of  claim 17 , wherein the grid is a pinhole grid and the light pattern produced by said projector is a grid of spot targets. 
     
     
       19. The in-vivo imaging system of  claim 12 , wherein said projector further comprises an illumination objective and said imaging system comprises an imaging objective. 
     
     
       20. The in-vivo imaging system of  claim 19 , wherein the illumination objective produces a depth of field of the grid of spot targets that is shorter than the depth of field of the imaging objective of the imaging system. 
     
     
       21. The in-vivo imaging system of  claim 19 , wherein the illumination objective produces a depth of field of the grid of spot targets that is larger than the depth of field of the imaging objective of the imaging system. 
     
     
       22. The in-vivo imaging system of  claim 12 , further comprising a selective reflector for reflecting the rays toward the subject. 
     
     
       23. The in-vivo imaging system of  claim 22 , wherein the projector is disposed to one side of the imaging system and said stage.